The cyclic interconversion of tyrosine residues of regulatory proteins between phosphorylated and unphosphorylated forms, or between nucleotidylated and unmodified forms, constitutes one of the most important mechanisms for regulation of key enzyme activities and of cell signaling networks. With the discovery that peroxynitrite is a normal product of nitric oxide metabolism in vivo, and that it is able to nitrate tyrosine residues of proteins, it was of interest to determine the effects of peroxynitrite on the activity off regulatory proteins. We reported last year that, in the presence of carbon dioxide, the peroxynitrite-mediated nitration of tyrosine residues in E. coli glutamine synthetase converts the enzyme to a form with regulatory properties similar to that obtained in vivo by adenylylation of a single tyrosine residue per subunit. In the meantime, it was shown that nitration of tyrosine residues in the adenylylated form of glutamine synthetase leads to complete inactivation of the enzyme. From a detailed analysis of the nitration patterns obtained following nitration of adenylylated and unadenylylated enzyme by means of proteolytic fragmentation, mass spectral measurements, and amino acid sequencing technologies, it was demonstrated that the tyrosine residue that participates in the adenylylation process is a prime target for nitration of the unadenylylated enzyme, but it is not nitrated in the adenylylated enzyme. The peroxynitrite-mediated inactivation of the adenylylated enzyme is due to nitration of a different tyrosine residue. These results support further the possibility that the toxicity of peroxynitrite may be attributable, at least in part, to the disruption of important regulatory functions.